Cystic fibrosis is caused by mutations in the gene for the anion channel CFTR. A link between CFTR, altered fluid transport and the persistent infections that cause most CF deaths has not been established. We will test the hypothesis that CF airways disease arises, at least in part, because of diminished or altered serous cell secretion. Our evidence is that CFTR is the only functional anion channel in the apical membranes of airway serous cells, so its loss will reduce rich serous cell secretion. We will directly test the hypothesis by comparing single gland secretions in airway tissues from CF and control humans. The series of experiments we propose have sufficient power to allow us to support or reject the serous cell dysfunction hypothesis. The proposal has 4 specific aims. The purpose of Aim 1 is to quantify and characterize airway submucosal gland secretions by using single gland monitoring to test hypotheses of gland function in pigs, sheep and cats. For individual glands, we measure the rate of mucus secretion, ion content, pH, and transepithelial potential difference and collect uncontaminated secretions to measure viscoelasticity, solids content, and secreted compounds such as lysozyme. Individual glands are labeled via dye injection and subsequent staining to provide single-gland, structure-function correlations. The goal of Aim 2 is to determine if gland secretions of CF subjects are diminished or altered by using the above methods to compare secretions from trachea and bronchi of normal control, disease control, and CF subjects, obtained following lung transplants. We project approximately 194 tissues will be acquired during the proposed 5-year grant period, consisting of 37 CF, 60 disease control, and 97 normal donor tissues. The design and interpretation of experiments in Aims 1 and 2 require a model of gland secretion, and the experimental results test the model. The purpose of Aim 3 is to develop a comprehensive model of ion transport for serous cells, using the Calu-3 cell model studied with open circuit Ussing chamber and pH stat studies. To link electrophysiological and fluid secretion studies, we have developed a novel method to study fluid secretion by epithelial cell sheets. In Aim 4, we will use this 'Virtual Gland' to study fluid secretion by Calu-3 cells and primary cultures of gland serous and gland mucous cells. The virtual gland operates in open circuit and can identify electrically silent mechanisms of fluid transport. The combination of methods will allow us to test the main hypothesis of altered gland function in CF, and will provide new information on airway gland function of use in other airway diseases.